Mycobacterium tuberculosis (Mtb) is an intracellular pathogen and a key to its success is the ability to survive in the macrophage phagosome. For many microbes, phagocytosis by a macrophage kills, due to transfer to the lysosome or exposure to oxidative or nitrosative stresses. Mtb avoids these killing mechanisms by arresting phagosome maturation and residing in a relatively safe vacuole that bears similarities to the early endosome. The goal of this application is to explore the mechanisms of Mtb adaptation to the macrophage phagosome using functional genomic techniques including: transcriptional profiling of Mtb growing inside macrophages and novel transposon mutagenesis approaches for genetic screens to discover mutants defective in adaptation to the phagosome. The specific aims of this application are: 1) the development of fluorescent Mtb reporter strains that respond to specific environmental cues in vitro and in vivo. For this aim, genes regulated by phagosomal stimuli will be identified by microarray-based transcriptional profiling of Mtb growing intracellularly and in culture. Promoters from genes regulated by a specific stimulus both in vitro and in vivo will be used to express green fluorescent protein in Mtb. 2) Identification of Mtb mutants that are altered in their response to the phagosomal environment using a flow cytometry-based genetic screen. In this screen, the Mtb reporter strains will be transposon mutagenized and exposed to a stimulus of interest. Mutants with less fluorescence as compared to the wild type strain will be enriched by flow cytometry-based sorting. These loss-of-function mutants may have lesions in genes associated with sensing or modulating a response to the stimulus. This work will identify genes that Mtb uses to adapt to the macrophage phagosome. Some of these genes will represent targets for novel drugs or vaccine strategies. Additionally, the transcriptional profiles of intracellular Mtb gene expression will be a valuable resource for microbiologists in diverse fields of study. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is a microbe that requires intensive study. Worldwide, it is a leading cause of death by an infectious disease, there is no effective vaccine for adults, and current drug therapies are leading to the evolution of multidrug-resistant strains because of low compliance to prolonged antibiotic treatments. Tuberculosis is a highly infectious, airborne disease and given the relative ease of global travel, high rates of tuberculosis anywhere in the world represents a serious threat to the health of all Americans. A more complete understanding of tuberculosis biology is essential if we are to discover and implement novel therapies or vaccines.